LNA-i-miR-221 is a novel phosphorothioate backbone 13-mer locked nucleic acid oligonucleotide-targeting microRNA-221 designed for the treatment of human malignancies. To understand the pharmacokinetic properties of this new agent, including unbound/total clearance, we investigated the LNA-i-miR-221 protein binding in three different species, including rat (Sprague–Dawley), monkey (Cynomolgus), and human. To this end, we generated a suitable ultrafiltration method to study the binding of LNA-i-miR-221 to plasma proteins. We identified that the fraction of LNA-i-miR-221 (at concentration of 1 and 10 µM) bound to rat, monkey, and human plasma proteins was high and ranged from 98.2 to 99.05%. This high protein binding of LNA-i-miR-221 to plasma proteins in all the species tested translates into a pharmacokinetic advantage by preventing rapid renal clearance. The integration of these results into multiple allometric interspecies scaling methods was then used to draw inferences about LNA-i-miR-221 pharmacokinetics in humans, thereby providing a framework for definition of safe starting and escalation doses and moving towards a first human clinical trial of LNA-i-miR-221. A rising body of evidence indicates that microRNAs (miRNAs) can provide valuable therapeutic targets because of their potential to functionally regulate key oncogenic/tumor suppressor genes by simultaneous regulation of multiple-related pathways. Among several miRNAs, miR-221 has been widely investigated for its steady overexpression in a variety of solid and hematologic malignancies ,4, inducWe have already developed a full GLP-compliant ion-pair reversed-phase LC–MS/MS method for accurate quantification of LNA-i-miR-221 in preclinical animal models that was recently validated for a GLP rat study . This LNThe aim of safe animal dose extrapolation is to estimate appropriate human dose and exposure levels to ensure drug safety. This approach is required for the initial dose selection in the clinical phase. In cross-species extrapolation, several factors should be considered in order to limit prediction bias. These include pharmacological, physiological, and anatomical factors, and metabolic function . The relNon-compartmental analysis methods  were used for PK characterization of the plasma concentration data relative to i.v. administration to mice, rats, and monkeys. Terminal first-order elimination rates for LNA-i-miR-221 from plasma (lambda z), were calculated using non-compartmental log-linear regression of the decay curves for the respective species. Half-life (t1/2) was calculated by dividing 0.693 by the first-order elimination rate. Terminal Vd values were calculated by dividing the calculated systemic CL by the terminal half-life value. All these results are summarized in Cross-species regression using allometry was performed by linear regression of plasma clearance versus body weight (W). The equation used to relate the PK parameters (Y) to body weight (W) was as follows:u); the latter was calculated by correcting the measured CLvalues for the species-specific plasma protein-free fraction. Thus, a log-log regression of a PK parameter Y  will yield a y-intercept value of “a” and a slope of “b”: Estimations were made for both total (CLp) and unbound clearance (CLpThe size and magnitude of exponent (b) indicates how the physiological variable changes as a function of body weight . CL estiAccording to the “Guidelines on strategies to identify and mitigate risk for first-in-human and early clinical trials with investigational medicinal products” (EMEA/CHMP/SWP/28367/07) and identification of the no observed adverse effect level (NOAEL), the AUC at the expected safe/effective dose in humans was simulated based on the dose calculated by directly applying the NOAEL dose in rats (5 mg/kg/day) and the dose normalized by body surface area, calculated by applying a correction factor using Equation (2), which finally predicted a HED dose of 0.78 mg/kg/day.1/2 after i.v. bolus administration was relatively fast, generally within 1–1.5 h. For mouse PK, the first blood sample was collected 1.5 h after dosing and the initial rapid distribution phase was not accurately profiled. The extrapolated C0 value may therefore be less accurate in the mouse PK analysis. Additionally, he contribution of the initial distribution phase to the total AUC could not be correctly estimated. For this reason, the calculated mouse PK parameters were not used for the allometric scaling of human PK. The unbound clearance value was extrapolated from the total clearance using the following relationship:u is the unbound plasma clearance, fu is the unbound fraction and CLp the total plasma clearance.As summarized in The unbound clearance in humans was then extrapolated by direct allometric scaling of the calculated unbound CL values for the preclinical species.PK scaling was conducted using one or two species. The body weight values for human and preclinical species used in this prediction are reported in u). The term CL is generically used to refer to total or unbound drug concentration.The following two methods were used to predict human drug CL  Scaling of CL from two species was based on Equation (1a), in which the CLs of both species were plotted against body weight (W). Direct scaling of total CLp from two species (rat and monkey) is shown in rat–monkey has the same value as parameter “a” ) and “b” is fixed at 0.65.(ii) Tang et al.  suggesteScaling according to Tang et al. (as in Equation (3)) from rat and monkey CL is shown in For the prediction of human CLp using two-species scaling (option i), Equation (1a) was log transformed as Equation (1b).rat–monkey was calculated as discussed above.The CL value logarithm in the preclinical species was then plotted against the body weight logarithm, and the linear fit of the transformed data was used to extrapolate the values of parameters “a” and “b” in Equation (1a). The human CL was then estimated using Equation (1a) and the calculated “a” and “b” parameters. In two-species scaling according to Tang et al.  . A distinct CL prediction was obtained from each preclinical species specificity, linearity, range and lower limit of quantification (LOQ),(ii)within-batch precision and accuracy,(iii)matrix effect,(iv)stability in human, monkey, and rat plasma at 37 °C.A suitable ultrafiltration method for determination of the binding of LNA-i-miR-221 to human, monkey, and rat plasma proteins was developed at Citoxlab France . A previously validated LC/MS method for the quantification of LNA-i-miR-221 in rat plasma  was tranThe sample preparation was slightly adapted and optimized to the use of a high-resolution mass spectrometer  and the The unspecific binding was then assessed using an Amicon ultracentrifugation device. The results clearly showed high unspecific binding of the test item: 92% and 40% at 1 and 10 µM concentration, respectively (data not shown).To limit the unspecific binding of LNA-i-miR-221 to the Amicon vials, they were pre-coated using a customized miRNA of similar length. The unspecific binding dropped to 50% and 35% at 1 and 10 µM, respectively (data not shown). It was then decided to use this coating step to assess the protein binding of the LNA-i-miR-221 and to apply a correction factor to compensate for the reduced free LNA-i-miR-221 recovery .The fraction of the LNA-i-miR-221 bound to human, monkey, and rat plasma ranged between 98.2% and 99.05% . The vern = 3). This result is in good agreement with the range of bound values expected and demonstrates the reliability of the protocol used.The percentage of bound drug for propranolol, which is used as a reference control for highly bound drugs in human plasma, was 89.4% ± 0.75% in human plasma  human metabolism may differ significantly from that of animals;(ii) mechanisms of toxicity may not be known ;(iii) toxicity may be due to an unidentified metabolite, not the parent drug.Therefore, relying on PK models (based on the parent drug in plasma) to gauge starting doses would require multiple untested assumptions. In addition, uncertainties  have been shown to affect human pharmacological or toxicological outcomes.Even though the monkey is considered to be the most appropriate species because monkeys experience the same dose-limiting toxicity as humans, for nonclinical safety assessment of LNA-i-miR-221, species selection was based on the retention of the miR-221 sequence among different species [PS–ODNs show similar PK profiles in plasma and tissue among species independently of sequence ,23,24,25The inclusion of a plasma protein binding correction in the allometric approaches adopted did not significantly modify the prediction. This is clearly related to the very similar unbound fraction measured ex vivo (ultrafiltration) in rat, monkey, and human plasma. The fact that all the tested species showed a similar unbound fraction at both concentrations (1 and 10 µM) tested in the protein binding assay is encouraging in terms of expected similar PK behavior across the species, which is a basic assumption when allometric extrapolation is applied to predict CL.The HED dose and the relative exposure in humans was also predicted according to the “Committee for Medicinal Products for Human Use. Guideline on Strategies to Identify and Mitigate Risks for First-in-Human Clinical Trials with Investigational Medical Products” . The hummax [max correlates across nonclinical species with the mg/kg dose, and in such instances mg/kg scaling would be justified [Complement activation by systemically administered allometric scaling ASOs in humans is believed to be dependent upon Cmax . For somustified . An alteThe dose showing efficacy in the mouse animal model was 25 mg/kg . This doThis HED value should be compared directly to the MRSD. In practice, the MRSD for the clinical trial should be determined by dividing the HED derived from the animal NOAEL by the safety factor. The default safety factor that should normally be used is 10. If the human PAD (the pharmacologic HED) is lower than the MRSD, it may be appropriate to decrease the clinical starting dose for pragmatic or scientific reasons. This is not the case in our study, as the predicted PAD was slightly higher than the MRSD value even before applying the additional safety factor.2, which is considered to be a method suitable for predicting lower dose values . In this case a HED of 0.78 mg/kg was predicted versus 5.0 mg/kg NOAEL, which would have been the value if the scaling had been directly based on the mg/kg normalization.In the absence of a NOAEL in monkey, the rat NOAEL was used as it was the only available NOAEL value. As a more conservative approach, the HED was calculated based on mg/mAn alternative HED estimation approach can be derived from a PAD estimate. In this case, the pharmacological HED calculated was slightly higher than the MRSD value: 1.8 vs. 0.78 mg/kg/day. Conversely, the exposure in rats at the NOAEL dose (after 18 days of treatment) wasFinally, these results were integrated into multiple allometric interspecies scaling approaches which were used to draw inferences about LNA-i-miR-221 PK and safe dose in humans in the absence of other data in humans .u. According to the different allometric scaling approaches used, the predicted human exposure ranged from 6741 to 12,686 h·ng/mL based on total CL uncorrected for plasma protein binding, and from 6044 to 12,781 h·ng/mL based on unbound CL. The measured exposure (AUC) in rats at the NOAEL and the predicted human exposure at the HED seemed then to be similar.The calculated plasma exposure in humans, based on the LNA-i-miR-221 estimated human CL value  and on the NOAEL dose in rats, scaled to humans according to Equation (2), was 9849 h·ng/mL using CLp and 9321 h·ng/mL using CLpFor these allometric approaches we excluded PK data from the mouse study because the contribution of the initial distribution phase to the total AUC was probably underestimated, considering the absence of data for the first half hour after infusion. The predicted values, based on rat and monkey PK data, were similar and coherent. Considering the uncertainty about the mouse PK parameters, the effective AUC in the mouse efficacy model at the effective dose of 25 mg/kg was approximately 3226 h·ng/mL, a value around 2–3-fold lower than the exposure in humans at the HED . Conversely the PAD, scaled according to the approach suggested by the FDA guidelines, estimated an effective human dose approximately 2-fold higher than the HED, but still in the same order of magnitude.2EDTA was used. Information are reported in The study was approved by the Citoxlab France Ethical Committee (CIC48). LNA-i-miR-221 was provided by BioSpring GmbH . Pri-miR-221-222 (nucleotide sequence (5′ > 3′): GAG-AAT-GAA-AAA-TCG) was used as internal standard for the bioanalytical method and was provided by Exiqon . The 17-mer long single-strand oligonucleotide (nucleotide sequence (5′ > 3′): ACA-GAC-AGA-TGT-ATG-CA) used to saturate the non-specific absorption on the surface of ultrafiltration device was purchased from Eurofins Genomics. Formic acid (code 42375500) was purchased from Acros Organics. Ammonium hydroxide (code 221228) was purchased from Fluka. The phenol chloroform:isoamyl alcohol , hexafluoroiso-propyl alcohol (code 105228), triethylamine (code STBH2222), and phosphate-buffered saline (PBS) (code P447-100TAB) were purchased form Sigma-Aldrich. Methanol (code 451230000) was obtained from Carlo Erba. The Amicon Ultra-0.5 centrifugal filter, 30 kD ultrafiltration units  were purchased from Merck Millipore. About 50 mL of pooled frozen plasma from human, monkey, and rat (pool of donors) obtained from blood treated with KTo limit non-specific binding of the LNA-i-miR-221 onto the Amicon vials, they were coated with a 17-mer long single-strand custom-synthesized oligonucleotide. A 500 µL aliquot of coating working solution (10 µM) was added to each Amicon filter, which was capped and kept at room temperature for 30 min. The Amicon units were then centrifuged at 14,000 rpm for 10 min at room temperature, after which the insert was removed and the vials were vortexed for 1 min to coat the inner walls.n = 3). A 500 µL spiked plasma aliquot was loaded into the coated Amicon ultrafiltration vials (n = 3). The vials were centrifuged at 14,000 rpm for 10 min at room temperature. After removing the insert, a 360 µL aliquot of filtrate was transferred into an Eppendorf vial containing 40 µL of human, monkey, or rat plasma. All samples were processed in the same way as the calibration curve samples. The percentage of unbound LNA-i-miR-221 was calculated as the ratio between its concentration in the filtered extract and its concentration in the non-filtered extract multiplied by 100. The percentage of protein-bound LNA-i-miR-221 was calculated by subtracting the unbound fraction from 100. Propranolol was used as a reference control. The incubations were performed using the Amicon ultrafiltration vials as for the LNA-i-miR-221, using a 1 µM concentration.Human, monkey, and rat plasma samples were spiked with the LNA-i-miR-221 at 1.0 and 10 µM . After brief vortexing, the samples were incubated in a thermostatic shaking plate at 37 °C for 30 min to allow the test item to bind to the plasma proteins and reach equilibrium. Afterwards, for each LNA-i-miR-221 concentration tested, a 40 µL plasma aliquot was removed and added to a 360 µL aliquot of PBS (n = 3). A second 500 µL aliquot was loaded into the Amicon ultrafiltration vials (n = 3). The vials were centrifuged at 14,000 rpm for 10 min at room temperature. After removing the insert, 360 µL of filtrate was transferred into an Eppendorf vial and added to a 40 µL aliquot of rat plasma. All samples were then processed in the same way as the calibration curve samples. The percentage of test compound non-specific binding was calculated as the ratio between the test item concentration in the filtered extract and its concentration in the non-filtered extract, multiplied by 100 [PBS was spiked with LNA-i-miR-221  in a glass tube. For each LNA-i-miR-221 concentration, a 360 µL aliquot was removed and added to a 40 µL aliquot of rat plasma  were calculated over the range of 10–2000 ng/ml, based on the calibration curve fitted by the weighted linear regression function (1/x2), with at least 75% of back-calculated concentrations within 15% accuracy. Analyte detection was done using an Orbitrap Q-Exactive (Thermo Scientific) apparatus operating in negative electrospray ionization mode. The HPLC separation was performed using a Halo C18 column . Analytes were eluted by applying a flow gradient separation (at 400 µL/minute), starting from 80% of phase A and 20% of phase B (where A was Milli-Q water/Hexafluoroisopropanol (HFIP)/trimethylamine (TEA) 100/4/0.2 v/v/v and B was acetonitrile/HFIP/TEA 100/4/0.2 v/v/v) and increasing the percentage of B up to 80% over 2 min. The composition of mobile phase was then kept constant for an additional 1.6 min and then set back to the initial conditions, leaving the column to re-equilibrate for 2.4 min before a new sample injection. The mass spectrometer was operated in full MS scan between 700 and 900 m/z and was set at 70,000 FWHM mass resolution power. LNA-i-miR-221 was detected following the m/z 875.0175 signal. Its retention time was approximately 2.65 min, while the internal standard eluted at approximately 2.75 min. A 400 µL sample matrix of plasma/PBS 10/90 v/v was extracted with 100 µL of phenol/chloroform/isoamyl alcohol 25/24/1 v/v/v solvent. The organic fraction was collected and evaporated under heated nitrogen gas (at 35 °C). After evaporation, the processed sample was re-suspended using 80 µL of the mobile phase mix (A/B = 80/20) before LC-MS analysis.The bioanalytical method adopted was based on a previously published method , but wit2 and the human equivalent dose (corresponding to the NOAEL in rats) was predicted to be 0.78 mg/kg/day. The calculated plasma exposure in humans, based on the estimated human LNA-i-miR-221 CL value, is 9849 h·ng/mL using CLp and 9321 h·ng/mL using CLpu. According to the different allometric scaling approaches used, the predicted exposure in humans ranges from 6741 to 12,686 h·ng/mL when based on total CL uncorrected for plasma protein binding, and from 6044 to 12,781 h·ng/mL when based on unbound CL. Finally, the human extrapolated PAD value based on the mouse effective dose and mouse BSA-CF was 1.82 mg/kg. The project flow and the results are summarized in Definition of an initial dose for first-in-human studies is complex, and a case-by-case approach may be appropriate depending on the product category  or new biological entities (NBE)). A conservative and consistent approach is required because safety is the most important aspect from a regulatory perspective. For LNA–oligonucleotides, few examples of human PK prediction are available in the literature. The objective of our investigation was to apply and compare different allometric approaches for predicting the CL and exposure of the novel drug candidate LNA-i-miR-221 in humans and to draw inferences about safe human plasma levels in the absence of human data. This was accomplished by applying multiple allometric interspecies scaling approaches to predict the CL of LNA-i-miR-221 oligonucleotides in humans from animal data. In the absence of monkey NOAEL values, the rat NOAEL of LNA-i-miR-221 was used. The HED was calculated as mg/mThe optimal PK of LNA-i-miR-221 relies on a large systemic Vd and broad tissue penetration. The plasma protein binding offers the additional advantage of preventing rapid renal clearance. Our evidence that all tested species showed similar unbound fraction values is reassuring in terms of expected similar interspecies PK behavior, which is a basic assumption when allometric extrapolation is used to predict human CL. The NOAEL and the protein binding results led us to predict the safe HED and the relative exposure in humans, and to provide further valuable information on LNA-i-miR-221 PK to be confirmed in the ongoing first-in-human study.LNA-i-miR-221 and its uses are patented: European Patent No. 2943570; United States Patent No. US 9,404,111,B2; Italian Patent No. 0001429326.


Microcystis and Nostoc in mass transfer and instantaneous temperature shock were explored by noninvasive microtest technology (NMT) after a series of pretreatments, to obtain the external layers retained or stripped samples. The results showed no statistical influence on photosynthetic activity between retained and stripped samples in both Microcystis and Nostoc. External-layer-retaining strains had higher net O2 effluxes than stripped strains. Moreover, the net NH4+ influx was significantly higher for the sheath retaining Nostoc than for the stripped sample, indicating that external layers might be an important feature driving mass transfer in cyanobacteria. However, the role of slime in NH4+ absorption was limited compared with that of sheath. In addition, external-layer-retaining strains exhibited a longer response time to instantaneous temperature shock, greater net O2 effluxes at a 4 °C shock and lower net O2 influx at a 35 °C shock, which were interpreted as reflecting a tolerance to temperature fluctuation over short time scales via a buffer function of external layers to stabilize cell activity, ameliorating the efficiency of photosynthesis and respiration. These results advance current knowledge regarding the external layers, especially the dense sheath, involved in the mass transfer in cyanobacteria, and provide new clues concerning the adaptive strategies of cyanobacteria under global climate changes.Groundwork on cyanobacterial external layers is crucial for an improved understanding of the persistent dominance of cyanobacteria in freshwaters. In this study, the role of two morphotypes of external layers in  Surface coats are widespread, surrounding microorganisms as well as some plant and animal cells. By comparing the surface coats of a variety of cell types, Bennett  proposedCyanobacteria possess an outermost structure around their cells, filaments or colonies, which differs from that observed in the bacterial group . The appThe existence of external layers  in cyanobacteria is generally considered necessary to infer its adaptive strategies in the natural environment . When thMicrocystis as an example, always occur in aggregates or colonies under natural conditions, but they tend to exist as single cells or a few paired cells in laboratory pure cultures [Current knowledge has shown that the combination of climate change, nutrient enrichment and cellular eco-physiological traits favor the dominance of cyanobacteria . Cyanobacultures . In genecultures . In thiscultures . A diffecultures . The forcultures ,22. In acultures . Climatecultures ,25. AlthMicrocystis species and two Nostoc species, which are surrounded by different types of external layers in freshwater sources. We used NMT to measure NH4+ and O2 net fluxes across the cyanobacterial cell surface, combined with instantaneous temperature shock to analyze their differences in tolerance. Our aims were to determine whether retention or removal of the external layers could modulate the mass transfer of cyanobacteria. The stress response to temperature of cyanobacteria was also explored. We believe that these are among the first and significant steps towards understanding the roles of various outermost layers in the adaptation and dominancy of cyanobacteria in the ever-changing environment.Noninvasive microtest technology (NMT) has been used in plant and algal research in recent years, and has provided new insights into net ionic and molecular fluxes by in vivo quantification ,27. It cMicrocystis species (M. aeruginosa FACHB-1338 and M. sp. FACHB-2427) and two Nostoc species (N. sp. FACHB-599 and FACHB-2009) were used in this study  to obtain stripped samples. Microcystis strains were subjected to ultrasound at 30 W for 60 s and then centrifuged at 12,000× g for 20 min (4 °C) to obtain stripped samples. Then, these external-layer-stripped cells were observed with India ink staining by light microscopy to check the sheath and/or slime.The first phase of the study was to obtain the external-layer-retaining and stripped samples of four species. Colonial l volume . Nostoc 4+ and O2 fluxes of external-layer-retaining and stripped samples of Nostoc and Microcystis were determined by NMT, to evaluate any changes after the removal of the sheath and/or slime. The external-layer-retaining and stripped samples of Nostoc and Microcystis were resuspended in BG11 N- medium, at a cell density of approximately 107 cells mL−1, followed by culturing under routine conditions for 24 h. The photosynthetic activity of these strains was measured, and, subsequently, 1 mL culture samples were placed in the middle of poly-L-lysine-pretreated coverslips (2 × 2 cm), in a measuring chamber consisting of a glass petri dish (35 mm). After the cells had settled on the coverslips, 5 mL NMT measuring solution was gradually added to the glass petri dish. NH4+ and O2 fluxes of all Nostoc and Microcystis samples were then monitored by NMT, respectively.In the second phase, NHM. sp. FACHB-2427 and N. sp. FACHB-2009 including two phenotypes, were used. The procedure involved the following: (i) the measuring chamber mentioned above was fixed in the center of another 100-mm glass petri dish; (ii) O2 fluxes were monitored at room temperature until the curve was stable; and (iii) water at different temperatures was rapidly added into the 100-mm glass petri dish separately, and an ice bath was applied at 4 °C, and a hot bath at 35 °C in the measuring chamber, respectively, with continuous O2 flux monitoring.In the third phase, to further understand the role of the sheath and/or slime in stress tolerance, an instantaneous temperature shock experiment was designed, in which All the NMT experiments described in this section were determined by measuring at least six similar samples separately, and each measurement was repeated three times at different positions of cell.Nostoc and Microcystis were observed with an inverted Olympus microscope  before and after staining with India ink.External-layer-retaining and stripped samples of v/Fm = (Fm-F0)/FmF [mF and 0F represent the maximum and minimum fluorescence values of the dark-adapted stage of PSII, and vF is the difference between them.In vivo chlorophyll fluorescence was measured with a phytoplankton analyzer . All strains were dark-adapted for at least 15 min, before measuring the fluorescence parameters . The maximum effective quantum yield of PSII was calculated according to the following equation: m-F0)/Fm , where F4+ and O2 were measured noninvasively using the noninvasive microtest technique . Prior to NH4+ flux determination, a prepulled and silanized microsensor  was first filled with a backfilling solution (100 mM NH4Cl) to a length of approximately 1 cm from the tip. The micropipette was front filled with 50–60 μm columns of selective liquid ion-exchange cocktails . An Ag/AgCl wire microsensor holder  was inserted in the back of the microsensor, to make electrical contact with the electrolyte solution. YG003-Y11 was used as the reference microsensor. For calibration of the NH4+ microsensor, we used measuring solution  with different concentrations of NH4+  to choose the qualified one with a Nernstian slope at 58 ± 5 mv/decade. The microsensor was then placed in the blank measuring solution for testing by X-10 until the NH4+ flux was near the baseline, at which time the microsensor can be used [Net fluxes of NH be used .2 flux measurement, the microsensor should be calibrated with measuring solution  containing different concentrations of O2 . Only when the Std Curve is between −2000~−9000 pA/mM can the microsensor be placed in the blank measuring solution for polarization for 1 h, until the net O2 flux is near baseline, at which time the microsensor can be used [To detect dissolved oxygen, the Pt/Ir polarographic oxygen microsensor  was used under -750-mV polarization voltage. A reference microsensor was also used to complete the circuit. Prior to O be used .4+ and O2 were determined by measuring six similar samples separately. The potential difference was obtained by moving the microelectrode repeatedly from one point to another, in a direction perpendicular to the surface of the individual cell (Polar X-10), and fluxes were calculated automatically by Fick’s law of diffusion: J = −D(dc/dx). The steady-state flux measurements were continuously recorded for 6–10 min, and each measurement was repeated three times at different positions of the cell.During formal measurement, the fluxes of NHhttp://www.youngerusa.com). For analyses of net NH4+ and O2 fluxes of external-layer-retaining and stripped samples of Nostoc and Microcystis, readings were averaged to obtain the net ionic and molecular steady fluxes for 6 min at each measurement position in each sample. The coefficient of variation (CV) was calculated as the standard deviation divided by the mean value. To determine the response time to instantaneous temperature shock, response curves of net O2 fluxes over 10 min were the first 5 points adjacent-averaging smoothed [The data obtained from NMT were exported as raw data and then converted into net fluxes by JCal V3.3 . The results of the experiment were analyzed by ANOVA, using Tukey’s post hoc test. All statistical analyses were carried out with Origin 9.0 . Differences were considered significant at Nostoc were characterized by the presence of a firm sheath surrounding the filaments. Within the sheath, the filaments were “randomly” loosely arranged, and they showed an irregularity of coiling in N. sp. FACHB-599, whereas filaments in N. sp. FACHB-2009 were more tightly packed , indicating that the low-frequency and power of the ultrasound applied in our study did not induce physiological deactivation of Nostoc and Microcystis. This result is consistent with the findings of Francko et al. [The extraction methods mainly consisted of physical processes, including ultrasound and centrifugation. An excessive intensity and duration of ultrasound is known to cause rapid and severe cell disruption and photosynthesis inhibition . In genel stress . The difo et al. , who cono et al.  reportedo et al.  proposedNostoc and Microcystis, the NMT technique was employed to monitor net NH4+ and O2 fluxes across the cell in real-time. This technique enables the observation of ion and molecule flux characteristics of biological phenomena, wherein positive values of flux data represent efflux and negative values influx [Nostoc and Microcystis mainly showed O2 effluxes in all external-layer-retaining and stripped samples, and the net O2 efflux in the retaining sample was significantly higher than its corresponding stripped sample . The average net O2 effluxes of strains with external layers in N. sp. FACHB-599 and FACHB-2009, M. aeruginosa FACHB-1338, and M. sp. FACHB-2427 were 4.45-fold, 1.74-fold, 1.88-fold and 2.14-fold higher than those of the corresponding stripped samples, respectively   , indicating that the external-layer-retaining strains showed little fluctuation and more stable NH4+ and O2 fluxes  a,b. The ectively e. Howeve > 0.05) c–e. More2 fluxes e. These tructure . The poounicells . Thus, t2 flux in N. sp. FACHB-2009 and M. sp. FACHB-2427 in external-layer-retaining and stripped samples were detected under instantaneous temperature shock at 4 °C and 35 °C, respectively  . The weighted average flux within the response time are shown in N. sp. FACHB-2009 and M. sp. FACHB-2427 showed greater net O2 effluxes than the stripped strains at 4 °C shock , probably because retaining strains can have a higher photosynthetic rate during cold adaptation. Compared with the sheathless strain of N. sp. FACHB-2009, the net O2 influx in the sheath strain was significantly lower at 35 °C shock. However, for M. sp. FACHB-2427 at 35 °C shock, the net O2 influx in the slime strain was slightly lower than the slimeless strain, although this difference was not significant . Crucially, the respiration rate of phytoplankton rises more rapidly with increased temperature than the photosynthetic rate, resulting in universal declines in the rate of carbon fixation with short-term increases in temperature [Nostoc colonies, can grow well when undergoing large temperature fluctuations on a daily basis [Based on the smoothed response curves of net O < 0.05) c–f. The ectively , suggestperature ,44. Throperature . This coly basis . Our finIncreasing concern about cyanobacterial blooms worldwide has motivated research on their external layers and EPS secretion . CyanobaTypically, the presence or absence of an external gelatinous layer is considered an important adaptation mechanism of cyanobacteria to their environment . As prevWith global climate changes, cyanobacterial blooms are predicted to expand. External layers of cyanobacteria can also function as a self-defense mechanism, to protect cells from climate changes. As proposed by Reynolds , externa2 effluxes than stripped strains, while the role of slime in NH4+ absorption is limited compared with that of sheath. Our instantaneous temperature shock experiments suggested that external-layer-retaining strains have temperature fluctuation tolerance over short time scales. We also deduced that the external layers, especially the dense sheath, are essential for this adaptation, and may have a buffer function and ameliorating impact on the efficiency of photosynthesis and photosynthesis-coupled respiration. These findings provide key insights into the dominance of cyanobacteria during climate changes.In conclusion, the external layer extraction methods employed in the present study can provide stripped strains, and had no statistical influence on their photosynthetic activity. Through NMT analysis, we show that external-layer-retaining strains have higher net O


Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB Acute lesions of the central nervous system (CNS), like traumatic brain injury and stroke, are major public health problems ,2,3,4. CMicroglial cells accumulate at the lesion site and play a key role in secondary lesion and the initiation of reparative mechanisms. During activation they undergo distinct morphological changes, proliferate, release cytokines and gain the ability of phagocytosis ,13. Micri/0-protein-coupled receptors -5-pentylbenzene-1,3-diol), a synthetic regioisomer of the phytocannabinoid cannabidiol with very low affinity to the classical cannabinoid receptors, mediates its action via a non-CB1, non-CB2 target. This receptor has first been described on blood vessels [Experiments with CB vessels ,41,42,43 vessels ,45. Vari vessels ,47,48,49 vessels ,51. The  vessels .1 or CB2 receptor antagonists, but were reduced by O-1918, an abn-CBD-sensitive receptor antagonist. Besides 2-AG abn-CBD caused neuroprotection and reduction in microglial accumulation at the injury site. These effects were abolished after depletion of microglial cells [Previously, in excitotoxically-lesioned organotypic hippocampal slice cultures (OHSCs), we demonstrated the mediation of the neuroprotective effects of 2-AG by abn-CBD-sensitive receptor . 2-AG imal cells ,54. Abn-al cells . In thisSince there is few data available on abn-CBD effects in astrocytes, we investigated the abn-CBD effects on a) glial production of pro-inflammatory mediators, and b) astrocytic scar formation with special regard to its temporal course in vitro.In the control groups of all examined cultures, only very low to non-measurable concentrations of nitrite, TNFα and IL-6 were detectable. Treatment with abn-CBD alone had no effect on the baseline production of NO, TNFα or IL-6 . LPS-tre2 knockout animals showed a significantly reduced response to LPS compared to the wildtype  or stroke are a common cause of persistent neurological failures, cognitive deficits and disability ,2. The s4-positive microglial cell was detected in isolated astrocytic cultures. In astrocytic-microglial cocultures, the microglial cells were partly clustered and partly localized between the astrocytes. Morphological signs for cell damage such as nuclear condensation or fragmentation were absent in all treatment groups of both cultures. In astrocytic-microglial cocultures IB4-positive microglial cells were found in each treatment group  and are converted to an activated state by LPS ,66,67. TIn our study, LPS treatment significantly stimulated NO, TNFα and IL-6 production in astrocytic-microglial cocultures. The LPS-stimulated production of NO, TNFα and IL-6 was much lower in isolated astrocytes. Our data is consistent with findings that quantitatively inferior microglial cells constituting the major part of pro-inflammatory micromilieu in CNS lesions ,73,74.LPS-induced IL-6 production in astrocytic-microglial cocultures was about two-fold higher than in isolated astrocytes. In contrast, LPS-induced NO and TNFα production differed between the cultures by a factor of about ten. This discrepancy might be explained by a relatively increased proportion of astrocytic involvement in IL-6 production. Whether astrocytes are able to express inducible NO synthases (iNOS), is a controversial issue. Cell culture experiments and immunohistochemical staining suggested that astrocytes cannot produce NO by inducing iNOS after stimulation with LPS, but may amplify microglial NO production due to cell–cell interaction ,75,76. I2 knockout.The basal production of NO, TNFα and IL-6 was low to not detectable in all cultures studied, and was not affected by abn-CBD. In cocultures of astrocytes and microglia, abn-CBD concentration-dependently reduced LPS-stimulated NO and TNFα production, while IL-6 production was not altered. This effect was not affected by CB2 receptor in abn-CBD-mediated effects. With regard to the vascular effects of abn-CBD, an orphan G-protein coupled abn-CBD-sensitive receptor was characterized [Since abn-CBD has no relevant affinity for the classic eCB receptors, there is some uncertainty regarding its molecular target structure ,77. Our cterized ,42,78. Scterized . The occcterized ,45. In tMicroglial iNOS induction and TNFα production are associated with neurotoxic effects in the context of CNS lesions. Excessive glial NO production may interfere with neuronal cell respiration, leading to excitotoxicity through the induction of neuronal and astrocytic glutamate release, while iNOS inhibition was neuroprotective ,17,79,80In cell culture experiments, the neurotoxicity of conditioned medium derived from LPS-activated BV2-microglia was reduced by abn-CBD . The red2 function, an overall altered immune response was observed in CB2 knockout cultures. Basal NO, TNFα and IL-6 levels were comparably low, but LPS-induced production of NO and TNFα was significantly lower, whereas LPS-induced IL-6 production did not differ. It is well accepted that the expression of CB2 receptors on microglial cells depends upon their activation state [2 receptor is involved in the modulation of inflammatory processes and microglial activity in the CNS [2 knockout cultures was unexpected, since previous work demonstrated that CB2 activation is associated with decreased microglial inflammation and neuroprotection in a mouse stroke model [2 function. In vitro, microglial expression of CB2 receptors is the subject of pathogen- or cytokine-specific regulation [2 knockout was also associated with a reduced inflammatory status, highlighting the model-dependent role of CB2 function [2 mimics the effects observed in knockout animals.While effects of abn-CBD were independent of CBon state . The CB2 the CNS . The redke model . This digulation ,88,89. Ifunction . A possiThe scratch-wound assay is widely used as a strongly reduced model for reactive astrogliosis and astrocytic scar formation. The lesion in this model is induced by the mechanical disruption of cell–cell contacts, as well as injury of cells in the wound area. Consecutive signals reach peripheral cells due to the syncytium-like cross-linking of astrocytes via gap-junctions and the paracrine secretion of cytokines ,81,91,92In comparison between isolated astrocytes and cocultures, no changes were observed at the time points investigated. Thus, in the present model, astrocyte wound closure does not appear to be a priori affected by the presence of microglial cells. In astrocytic-microglial cocultures, treatment with 10 μM abn-CBD significantly delayed the wound closure, while in isolated astrocytes no significant effect was detectable. Therefore, abn-CBD appears to influence the astrocyte response secondarily through its influence on microglial activity. In this context, the change in microglial cytokine production by abn-CBD may be an explanatory approach. This hypothesis is supported by experiments showing inhibition of outgrowth of astrocyte processes in the scratch-wound assay by blocking antibodies against TNFα . Quantification of scratch-wound assays is often done by microscopic measurement at one defined time point ,95,96,971-mediated psychotropic effect [2-independence of abn-CBD-mediated effects, the cultures with CB2 knockout showed a differentially reduced response to LPS. Further understanding of the underlying molecular mechanisms, will also contribute to a better understanding of the eCB system and neuroinflammatory cascades in secondary damage.In summary, our experiments show that abn-CBD is a modulator of glial cell activation by differentially altering the secretion of pro-inflammatory mediators. It affects the reorganization of astrocytes after mechanical lesion. This provides new explanations for the neuroprotective potential of a promising substance for pharmacological use. Thus, in contrast to other cannabinoids, there is no CBc effect ,98. WhilAll experiments involving animal material were performed in accordance with the directive 2010/63/EU of the European Parliament and the Council of the European Union (22.09.2010) and approved by local authorities of the State of Saxony-Anhalt (permission number: I11M18) protecting animals and regulating tissue collection used for scientific purposes.2 receptor knockout mice [2+ and Mg2+. Under stereomicroscopic observation, the meninges, olfactory bulb, cerebellum and brainstem were removed. The brains were rinsed three times briefly with HBSS without Ca2+ and Mg2+ before treatment with a solution of trypsin  and DNAse  in HBSS . After rinsing again with HBSS without Ca2+ and Mg2+, the brains were suspended using DNAse . The digestion was stopped by the addition of HBSS containing Ca2+ and Mg2+, and the suspension was centrifuged. The pellet was resuspended in Dulbecco’s modified Eagle’s medium  and the cell suspension transferred to Poly-L-Lysine -coated culture flasks. Suspension from four brains was used for one culture flask. Until further use, the cells were incubated at 37 °C and 5% CO2. After two days, the cell debris was removed by washing the cultures with HBSS, followed by the addition of fresh culture medium. Astrocytic-microglial cocultures were prepared from neonatal p0–1 C57BL/6 mice and corresponding CBout mice ,100. In v/v) fetal bovine serum , 1% (v/v) penicillin-streptomycin (Gibco BLR Life Technologies) and 0.1% (v/v) vitamin C (Sigma-Aldrich). Before use, the medium was heated (37 °C), pH adjusted to 7.4 and then sterile filtered. A change of culture medium was performed every other day. For cultivation, DMEM was used, containing 4.5 g/l glucose with the addition of 10%  solution (Biochrom) . The reaction was stopped by adding culture medium and the cell suspension was centrifuged. The supernatant was discarded, the cell pellet resuspended and passaged in fresh culture medium on two new PLL-coated culture flasks. After reaching confluence in the culture flasks, the cells were washed for 5 min at 20 °C with phosphate buffered saline  without CaTo obtain pure astrocytic cultures, microglia cells were depleted from the coculture by adding 10 μg/mL clodronate to the culture medium. The procedure was combined three times with the scheduled changes of culture medium. Thereafter, in the course of further cultivation, medium changes took place without the addition of clodronate. Clodronate at the concentration used leads to almost complete elimination of the microglial cells without affecting the activity or proliferation of astrocytes ,103,1042 receptor knockout animals were examined. At least four independent experiments were performed per cell culture and analyzed substance. In this context cultures of different animals are considered independent.All measurements were performed on primary astrocytic-microglial cocultures and on isolated astrocytic cultures. In addition to cultures from wildtype animals, cocultures and isolated astrocytes obtained from CB2). The supernatants were collected at the end of experiments and stored at −20 °C until further evaluation  and 50,000 cells were transferred into each compartment of a 24-well plate. The number of cells was ascertained by counting the cell suspension with a Neubauer counting chamber prior to appropriate dilution. After 24 h, the culture medium was changed and the cells were treated according to the protocol . Subsequaluation .For nitrite measurements, a Griess reagent optimized for use on cell culture supernatants was used  according to manufacturer’s instructions. Prior to measurement, standard concentrations were prepared as a dilution series of a 100 μM solution of sodium nitrite in culture medium at concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM and 0 μM. Subsequently, 50 μL of each sample and the standard concentrations were transferred to the compartments of a 96-well plate and treated with the same volume of Griess reagent. All measurements were done in duplicate. After 15 min, the absorbance at 540 nm was photometrically quantified by a microplate reader.2O2 and tetramethylbenzidine was added. This solution is the substrate of HRP, and the catalyzed reaction produces a blue reaction product, and the amount of depends on the quantity of HRP bound. The reaction was stopped by the addition of H2SO4 and the absorbances at 450 nm and 540 nm were immediately measured photometrically on the microplate reader. To correct optical errors of the 96-well plate, the measured values at 540 nm were subtracted from the values at 450 nm. Cytokine measurements were carried out by sandwich enzyme-linked immunosorbent assay (ELISA) . CommercScratch-wound assays were performed with isolated astrocytic cultures and astrocytic-microglial cocultures obtained from C57BL/6 wildtype mice. After the confluent growth in the culture flask, 500,000 cells were transferred into the compartments of a 6-well plate. The number of cells was ensured by counting the cell suspension with a Neubauer counting chamber prior to appropriate dilution. In each experiment three wells of the plate were colonized with astrocytic-microglial cocultures and the remaining three with isolated astrocytic cultures. Subsequently, the cultures were incubated for 24 h until adherence and confluence were achieved. A central, vertical and straight scratch was placed using a 10 μL pipette tip. The culture medium was replaced with fresh medium to which the respective substances were added according to the protocol. The cells were treated with abn-CBD at concentrations of 1 μM and 10 μM or methylacetate corresponding to the amount of solvent in 10 μM abn-CBD treated group b.2. The examined positions were defined, so that in each compartment of the plate three overlap-free areas were imaged from the central area of the scratch. Furthermore, an additional representative Wound closure was observed with a Leica live cell microscopy system by using a 20× phase contrast objective, while the cells were incubated constantly at 37 °C and 5% COA total of ten independent experiments were performed. In this context, cultures of different animals are considered independent. Positions where the wound margins were not completely visible initially or at a later time point were excluded from analysis for all time points. However, from each treatment group the images from at least eight independent experiments were evaluated. The MatLab script TScratch was used to determine the percentage image area that was not covered by cells . Since a2 greater than 0.98 was achieved for each standard curve, linear regression proved to be the appropriate basis for calculations. For quantification of cytokine measurements mean values from duplicate measurements were formed. Based on the measured standard concentrations, the best possible standard curve was determined by four-parametric logistic regression, and the unknown values were interpolated. The values of IL-6 measurements were multiplied according to the 1:10 dilution. Regression of the standard curve and interpolation of the unknown values was performed with a MatLab script. The groups were tested for normal distribution by Shapiro-Wilks test. Since the existence of normal distribution was confirmed in each group, the calculated nitrite or cytokine concentrations were analyzed for differences with one-way ANOVA and subsequent Bonferroni’s post-test. For linear regression, interpolation and statistical analysis the software GraphPad Prism 5 was used.For quantification of nitrite measurements mean values from duplicate measurements were formed. The standard curve was created by linear regression and the unknown values were interpolated. Since an RFor quantification of the scratch-wound assay the mean values were calculated from three values per well and time point. Progressive wound closure was quantified by the cumulative change of the cell-free image area compared to the cell-free image area at time point zero in the respective well. In addition, the respective changes in cell-free image area at the 6-h intervals 0 h–6 h, 6 h–12 h, 12 h–18 h and 18 h–24 h, were analyzed to characterize the temporal dynamics of wound healing and treatment effects. The groups were tested for normal distribution by the Shapiro-Wilks test. Since the existence of normal distribution was confirmed in each group, statistical analysis was performed with one-way ANOVA and Bonferroni’s post-test using GraphPad Prism 5.Griffonia simplicifolia isolectin B4  was performed . The cells were washed again with PBS-Triton  and then incubated with 4′,6-diamino-2-ohenylindole . Sections were washed with aqua destillata , mounted with DAKO fluorescent mounting medium (Agilent) and analyzed by confocal laser scanning microscopy (Leica). Cellular nuclei, astrocytes and microglial cells were visualized using monochromatic light, emission filters with the specified wavelengths and 40× objective. Exploratory immunohistochemistry was performed on wildtype astrocytic-microglial cocultures and isolated astrocytes. Treatment of the cells and seeding on coverslips proceeded exactly as for cytokine and nitrite measurement .. Cells


Electron microscopy revealed more abnormalities which involve the retinal pigment epithelium (RPE) and Bruch’s membrane, outer and inner layers, vascular changes, deposits of amorphous materials, and accumulation of lipids or lipofuscins. Age-related miRNAs, miR-27a-3p (p < 0.01), miR-27b-3p (p < 0.05), and miR-20a-5p (p < 0.05) were significantly up-regulated in aged male mice compared to the controls, whereas miR-20b-5p was significantly down-regulated in aged male (p < 0.05) and female mice (p < 0.05) compared to the respective controls. miR-27a-3p  and miR-27b  were significantly up-regulated in aged male mice vs. aged female mice, whereas miR-20b-5p  was significantly down-regulated in aged male mice vs. aged female mice. Interestingly, miR-27a-3p, miR-27b-3p, miR-20a-5p, and miR-20b-5p expressions significantly correlated with the thickness of the retinal PS layer (p < 0.01), retinal outer layers (p < 0.01), and Bruch’s membrane (p < 0.01). Our results showed that biological sex can influence the structure and function of the retina upon aging, suggesting that this difference may be underlined by the dysregulation of age-related mi-RNAs.Age and gender are two important factors that may influence the function and structure of the retina and its susceptibility to retinal diseases. The aim of this study was to delineate the influence that biological sex and age exert on the retinal structural and ultrastructural changes in mice and to identify the age-related miRNA dysregulation profiles in the retina by gender. Experiments were undertaken on male and female Balb/c aged 24 months (approximately 75–85 years in humans) compared to the control (3 months). The retinas were analyzed by histology, transmission electron microscopy, and age-related miRNA expression profile analysis. Retinas of both sexes showed a steady decline in retinal thickness as follows: photoreceptor (PS) and outer layers ( The structural and functional physiological evolution of the retina with age can be influenced by biological sex and susceptibility to retinal diseases . There aThe anatomical and physiological differences of the retina related to gender are reflected in the associated pathologies . Aging iMicroRNAs (miRNAs), a class of short noncoding RNAs, have been identified that are up- or down-regulated during mammalian aging , but no Limited results are available regarding the ultrastructural retinal changes upon human aging and in age-related retinal diseases  or in prAll experimental procedures were conducted in compliance with the European and national regulations for the care and use of animals for scientific purposes. Ethics approval was obtained from the Ethics Committee for research of the Vasile Goldis Western University of Arad .n = 10/each group/sex). The life span of laboratory mice ranges from 2 to 2.5 years; thus, 24 months approximate 75–85 years of age for a human  and 24 months  of age, respectively  was added to PBS . To increase the efficiency of perfusion, heparin . The light microscopy images were acquired using an Olympus BX43 microscope, with an XC30 CCD and cellSens Dimension Imaging Software .The eye samples were prefixed in 2.7% glutaraldehyde solution  in 0.1 M phosphate buffer, then washed in 0.15 M phosphate buffer (pH 7.2), and postfixed in 2% osmic acid solution  in 0.15 M phosphate buffer. Dehydration was performed in acetone, followed by embedding in the epoxy resin (Epon 812). Thin sections of 70 nm thickness were cut on a Leica EM UC7 ultramicrotome . The double staining of thin sections on grids was performed with solutions of uranyl acetate and lead citrate. The sections were imaged under a TEM  at 80 kV. Data acquisition was performed with a MegaView III CCD using iTEM SIS software .For each mouse, the thickness of the retinal layers  were measured on ten different histological sections taken from the superior and inferior retinas  every 300 µm from the optic nerve head (ONH) using CellSens Dimension Imaging Software .n = 50 for each group) acquired at the same magnification  using iTEM SIS software  and exported in Excel format for statistical analysis.The morphometry on the electron microscopy images was performed for the equatorial retina. The thickness of Bruch’s membrane was measured at three different points on the images (N = 10 per group) by using the MiRNeasy Mini Kit , according to the manufacturer’s instructions. An appropriate volume of QIAzol Lysis Reagent  was used for tissue homogenization, and the miRNA isolation efficiency was monitored by adding Syn-cel-miR-39 miScript miRNA Mimic 5 nM  to each sample before RNA purification. RNA quality and concentration were determined by a NanoDrop 2000c spectrophotometer . Mature miRNAs were converted into cDNA by reverse transcription performed by using the MiScript II Reverse Transcription Kit  and a Gene Amp PCR System 9,700 . The levels of eight miRNAs , which were previously shown to be time-dependently dysregulated in the diabetic retina  and then expressed as the fold regulation. In particular, the fold up-regulation was equal to the fold change value, whereas the fold down-regulation was calculated as the negative inverse of the fold change value . Differences were considered statistically significant for p values <0.05.Values are expressed as means ± SD of p < 0.01 aged male vs. control; p < 0.05 aged female vs. control). The thickness of the INL was reduced 1.27-fold for the males and 1.04 times for the females, whereas the IPL decreased 1.25-fold for males and 1.05-fold for females. The retinal pigment epithelium (RPE) and retinal ganglion cell layer (GGL) reduced approximately onefold in older mice compared to young ones and were similar for both sexes.The aging-induced changes in the entire retinal structure were analyzed. Representative retinal cross sections obtained from male and female mice are shown in The control retinal pigment epithelial cells appeared with a normal aspect, separated from the choroid by Bruch’s membrane (BM), showing cytoplasmic melanin granules, mitochondria, and a regular nucleus , 3. The For both controls, the photoreceptor discs were intact and properly aligned and were perpendicular to the photoreceptor axis. In aging states, impairment of the photoreceptor outer segments was observed . The rodMoreover, in old mice, we found photoreceptor nuclei of variable shapes, sizes, and chromatin densities, and some had pyknotic nuclei and empty spaces, indicating nuclear loss. Swelling of mitochondria was observed mainly in aged male photoreceptor cells  comparedThe inner nuclear layer (INL) of the controls had a normal aspect, was abundant in bipolar cells, and ensheathed by processes of Müller cells. Electron micrographs of both aged retinas showed an extensive network of cytoplasmic processes of Müller cell wrapping around other cells and penetrating the neighboring inner plexiform layer (IPL) . AdditioThe inner plexiform layer of the controls clearly showed different types of processes . The preThe ganglion layer was also affected with age . The nump < 0.01), miR-27b-3p , and miR-20a-5p  were significantly up-regulated in aged male mice compared to control male mice, whereas miR-20b-5p was significantly down-regulated  (p < 0.05) (p < 0.01) compared to that in aged females as well as miR-27b-3p  and miR-20a-5p , whereas miR-20b-5p was significantly down-regulated in aged males  compared to that in aged females  were not dysregulated between the control and aged experimental groups . Convers < 0.05) . Moreove < 0.05) . miR-27a females .p < 0.01), whereas miR-20b-5p expression was positively correlated with PS, ONL, and OPL thickness (p < 0.01) (p < 0.01), whereas it showed a negative correlation with miR-20b-5p expression (p < 0.01)  , 9. In c < 0.01) .Age and gender are two important factors that may influence the function and structure of the retina. Other results did not show a significant difference between the thickness of the retinal layers and age, by sex in groups of Sprague Dawley rats aged 1, 2, 6, and 10 months , which hAge-related macular degeneration (AMD) in humans is associated with progressive degeneration of the retinal pigment epithelium (RPE) cell layer and photoreceptor cells. Because photoreceptors depend for their maintenance on RPE, it was essential to analyze the involvement of RPE in changes related to aging. The senescence-accelerated mouse (SAM) strain was shown to exhibit changes only in Bruch’s membrane and RPE . ElectroAging is also associated with accumulation of retinal lipids and lipofuscins, subsequently contributing to AMD , to bestThe lipid source of photoreceptors is plasma lipids, and their transfer is facilitated by the RPE and Müller cells. Moreover, the receptors for LDL were highlighted on the RPE  connecteThe outer segments of the photoreceptors are composed of infolded plasma membrane discs with photopigments that are properly aligned, and establish an anatomical association with the RPE . SeveralA proposed mechanism that could trigger the degenerative process in aging is primarily focused on the outer segments of the photoreceptors, followed by the inner segments, and finally on the cell body depletion from the ONL . A deepeThe INL contain three types of neurons , a macroglia, and a Müller cell, which span the entire thickness of the retina, and they are in anatomical and functional contact with all retinal neurons. The phenotype changes of the Müller glial cells are highlighted by the presence of an extensive network of cytoplasmic processes wrapping around other cells and penetrating the neighboring inner plexiform layer (IPL). The electron microscopy observations may be correlated with data regarding Müller cell resistance in ischemia, anoxia, or hypoglycemia  to the dThe ultrastructural aspect of the ganglionar layer was affected with age with a decline in the number of ganglion cells, as was shown by other aging studies  or otherMicroRNAs (miRNAs) have been established as significant regulators of senescence and cell aging  by inducFurther evidence supporting a dysregulation of age-related miRNAs in the retinal structure and function was shown by our correlation analysis, showing a significant association between miRNA expression levels and the thicknesses of PS, ONL, OPL, and Bruch’s membrane. Although miR-27a-3p, miR-27b-3p, miR-20a-5p, and miR-20b-5p were shown to be dysregulated in retinal derangement induced by diabetes , these mInterestingly, gender-related miRNAs have been previously reported to be dysregulated in neurological disorders: miR-27a was up-regulated in epilepsy and autoimmune encephalomyelitis ; higher


Streptococcus pneumoniae infection. Employing a mouse model of pneumococcal infection, it has been shown that passively administered human wild-type CRP protects mice against infection, provided that CRP is injected into mice within two hours of administering pneumococci. Engineered CRP (E-CRP) molecules have been reported recently; unlike wild-type CRP, passively administered E-CRP protected mice against infection even when E-CRP was injected into mice after twelve hours of administering pneumococci. The current study was aimed at comparing the protective capacity of E-CRP with that of an antibiotic clarithromycin. We established a mouse model of pneumococcal infection in which both E-CRP and clarithromycin, when used alone, provided minimal but equal protection against infection. In this model, the combination of E-CRP and clarithromycin drastically reduced bacteremia and increased survival of mice when compared to the protective effects of either E-CRP or clarithromycin alone. E-CRP was more effective in reducing bacteremia in mice treated with clarithromycin than in untreated mice. Also, there was 90% reduction in antibiotic dosing by including E-CRP in the antibiotic-treatment for maximal protection of infected mice. These findings provide an example of cooperation between the innate immune system and molecules that prevent multiplication of bacteria, and that should be exploited to develop novel combination therapies for infections against multidrug-resistant pneumococci. The reduction in antibiotic dosing by including E-CRP in the combination therapy might also resolve the problem of developing antibiotic resistance.C-reactive protein (CRP) is a component of innate immunity. The concentration of CRP in serum increases in microbial infections including  Streptococcus pneumoniae (in vivo is due to the ability of ligand-complexed CRP to activate the complement system (C-reactive protein (CRP) is a critical host defense molecule of the innate immune system , 2. CRP eumoniae . Once boeumoniae –9. Expereumoniae –15. It ht system –18. Howet system .40 and Glu42 are present in the intrinsically disordered region of CRP. Glu42 is also a part of the inter-subunit contact region. Phe66, Thr76 and Glu81 form the PCh-binding site of CRP  according to manufacturer’s instructions. The concentration of endotoxin in CRP preparations was determined by using the Limulus Amebocyte Lysate kit QCL-1000 (Lonza). Purified CRP was stored at 4°C and used within a week for mouse protection experiments.The cDNAs for E-CRP-1 (E42Q/F66A/T76Y/E81A mutant CRP) and E-CRP-2 (Y40F/E42Q mutant CRP) were constructed and expressed in CHO cells using the ExpiCHO Expression System (Thermo Fisher Scientific), as described earlier . Purific earlier . E-CRP-2Streptococcus pneumoniae type 3, strain WU2, was obtained as a gift from Dr. David Briles . Pneumococci were made virulent by sequential i.v. passages in mice and were stored in aliquots at −80°C, as described previously (8 cfu/ml based on the absorbance of the resuspension at 600 nm (A600 = 1.00 = 1.2 × 109 cfu/ml). Within 2 h, 100 µl (3.5 × 107 cfu) of pneumococci suspension was injected into each mouse, as reported previously  were brought up and maintained according to protocols approved by the University Committee on Animal Care. Mice were 8–10 weeks old when used in experiments.in vivo experiments, clarithromycin (50 mg/ml) was diluted in acetone at 40, 4.0, 0.4 and 0.04 mg/ml, and 50 µl of each concentration was injected i.v. into mouse, four times, at 12, 36, 60 and 84 h after the administration of pneumococci. Thus, the final amount of clarithromycin in each group of mice was 2, 0.2, 0.02 and 0.002 mg per mouse per injection. As shown in The antibiotic clarithromycin  was reconstituted in acetone at a concentration of 50 mg/ml and stored at 4°C for a maximum of 5 days. To determine the experimental dose for 7 cfu of pneumococci, as described earlier  was diluted in normal saline to a final concentration of 0.2 mg/ml, and 100 µl was injected i.v. per mouse at 13, 36, 60 and 84 h after the administration of pneumococci, to achieve a final dose of 0.02 mg of clarithromycin per mouse per injection. Survival of mice was recorded three times per day for 7 days. To determine bacteremia (cfu/ml) in the surviving mice, blood was collected daily for 5 days from the tip of the tail vein, diluted in normal saline, and plated on sheep blood agar for colony counting. The bacteremia value for dead mice was recorded as 109 cfu/ml because mice died when the bacteremia exceeded 108 cfu/ml.Two separate protection experiments were performed using 25 μg of purified WT CRP, E-CRP-1 and E-CRP-2, and 3.5 × 10 earlier , 15, 21.p-values for the differences in the survival curves among various groups, the survival curves were compared using the software’s Logrank (Mantel-Cox) test. The scatter plots of the bacteremia data and the median bacteremia value for each group were generated using the GraphPad Prism 4 software. Bacteremia values of 0–100 were plotted as 100 and bacteremia values of >108 were plotted as 109. To determine p-values for the differences in bacteremia among various groups at each time point, scatter plots were compared using the software’s Mann-Whitney test. The software’s Mann-Whitney test included all the dots in the scatter plots and not just the median values for each time point.Survival curves were generated using the GraphPad Prism 4 software. To determine As shown in 4 cfu/ml in mice treated with both E-CRP-1 and clarithromycin. The median bacteremia was maintained at the reduced level from the beginning to the end of the experiment.The protective effects of E-CRP-1 (group B) and clarithromycin (group C) on the survival of mice, when used alone, were due to significant decrease in bacteremia , and claOverall, based on the statistical analyses of the survival curves Figure 2As shown in 5 cfu/ml in mice treated with both E-CRP-2 and clarithromycin. The median bacteremia was maintained at the reduced level from the beginning to the end of the experiment.The protective effects of E-CRP-2 (group B) and clarithromycin (group C) on the survival of mice, when used alone, were due to significant decrease in bacteremia , and claOverall, based on the statistical analyses of the survival curves Figure 4E-CRP molecules capable of binding to factor H recruited on the surface of pneumococci have been shown to protect mice against late-stage pneumococcal infection . AntibioBoth E-CRP and clarithromycin decrease bacteremia, but via different mechanisms. E-CRP-mediated decrease in bacteremia is due to the activation of the complement system component of innate immunity. Clarithromycin is a macrolide antibiotic that has bacteriostatic action against gram-positive bacteria and some gram-negative bacteria including anaerobes. Clarithromycin is believed to function by binding to the ribosome within the microorganism and inhibiting protein synthesis, and thus inhibiting bacterial growth –29. A poA single dose of E-CRP (25 μg) combined with a tiny amount of clarithromycin (0.02 mg) was the best prescription among all others in this study and in a previously published study  for nearCombination therapies using antimicrobials with different mechanisms of action are used to treat infections against antibiotic-resistant pneumococci and, at the same time, are formulated to prevent the spread of the resistance –41. CombWe conclude that the efficiency of the innate immune system is enhanced in the presence of antibiotics. Our findings provide another example of cooperation between the innate immune system and molecules that prevent multiplication of bacteria, and that should be exploited to develop novel combination therapies for infections against antibiotic-resistant pneumococci. The reduction in antibiotic dosing by the strategy to include E-CRP in the combination therapy using antibiotics might also resolve the problem of developing and spreading antibiotic resistance.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by University Committee on Animal Care, ETSU.DN and SS performed the experiments. AA conceived and designed the experiments. DN, SS, and AA analyzed the data. AA wrote the manuscript with input from DN. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health grants AR068787, AI117730. and AI151561.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To examine the association between meteorological factors  and transmission capacity of COVID-19.0) of COVID-19. We determined the correlations between meteorological factors and R0 of COVID-19 using multiple linear regression models and meta-analysis. We further validated our results using a susceptible-exposed-infectious-recovered (SEIR) metapopulation model to simulate the changes of daily cases of COVID-19 in China under different temperatures and relative humidity conditions.We collected daily numbers of COVID-19 cases in 202 locations in 8 countries. We matched meteorological data from the NOAA National Centers for Environmental Information. We used a time-frequency approach to examine the possible association between meteorological conditions and basic reproductive number (R0 of COVID-19 (meta p = 0.446). Also, relative humidity (meta p = 0.215), wind speed (meta p = 0.986), and ultraviolet (UV) radiation (meta p = 0.491) were not significantly associated with R0 either. The SEIR model in China showed that with a wide range of meteorological conditions, the number of COVID-19 confirmed cases would not change substantially.Temperature did not exhibit significant association with R0 of COVID-19. Warmer weather alone seems unlikely to reduce the COVID-19 transmission.Meteorological conditions did not have statistically significant associations with the R  •0.Temperature did not exhibit significant associations with COVID-19's R•0.Other meteorological factors weren't significantly associated with COVID-19's R•It's unlikely to depend on warmer weather to reduce the COVID-19 transmission. It was first identified in December of 2019 in Wuhan, China, and has since become an ongoing pandemic. It has impeded global development and stressed health care delivery systems worldwide . The nume.g., temperature, relative humidity) . For example, it was reported that the transmission of SARS can be influenced by meteorological factors (umidity) . Given tumidity) . Severalumidity) . The finumidity) . Other sumidity) . The difTherefore, this study aims to comprehensively examine the associations between meteorological factors  and COVID-19 transmission at a global scale. Further, we validated our results using a susceptible-exposed-infectious-recovered (SEIR) metapopulation model to simulate changes of daily cases of COVID-19 in China under different temperatures and relative humidity conditions.22.1From the databases compiled by 0) of COVID-19. In brief, R0 represents the expected number of secondary cases produced by an initial infectious individual in a completely susceptible population  there should be consecutive case days lasting at least four days, and 2) the number of newly confirmed cases should not equal to one in any of these four days, then 3) the first day of the aforementioned dates was chosen as the first case-day in the study regions. In each location, a total of 18 days  were used to estimate R0.All these locations have cumulative number of cases over 50 to enable estimation of the basic reproductive number  data were extracted from the Dutch-Finnish Ozone Monitoring Instrument (OMI) Level 2 UV irradiance products with version 003 (OMUVB V003) at 13 km × 24 km resolution. OMI is a nadir-viewing spectrometer aboard the NASA Aura satellite covering UV wavelength from 270 to 380 nm. The average of EDD values from OMI pixels matched within these locations was assigned as the daily mean EDD level for the corresponding locations.2.3e.g. COVID-19 cases and temperature) were used to implement wavelet coherency analysis . Then we used one-way ANOVA analysis to test the differences between the daily confirmed cases of COVID-19 under different meteorological conditions.Finally, to validate the observed associations of meteorological factors with the COVID-19 transmission, we constructed a susceptible-exposed-infectious-recovered (SEIR) model  using Chinese data. We chose China because more detailed meteorological data and case information were available only in the Chinese cities. Specifically, when a susceptible (S) person encounters a COVID-19 case, this person is likely to be infected by the virus (E) and progress to the infected phase (I), and the person may appear to have obvious symptoms and be subsequently diagnosed. The patients might be treated (T), or they have been showing asymptomatic infection and stay in sub-clinical stage (U), and then most of them will join the rehabilitation group . The flow diagram for the model appears in Fig. S1 and the formula for the model are shown below, of which βThe detailed formula in the SEIR model are as the following:p value of less than 0.05 was considered to indicate statistical significance.All calculations were completed in R software version 3.6.1  and MATLAB R2019b. A 33.10 of COVID-19 were  in 202 districts. The top three locations with highest R0 were Hamburg and Hessen in Germany and New York in the US. The average temperature was 6.7 °C, with a range of −23.0 °C to 29.6 °C and the median ± interquartile range for temperature in these locations were shown in Supplemental Table 1.Among the 202 locations, the mean ± standard deviation and range of R0 of COVID-19 in these locations exhibited different spatial patterns  . 3.30 of COVID-19 , relative humidity (meta p = 0.215), and wind speed (meta p = 0.986) were not significantly associated with R0  either  either .Fig. 6Th3.4p = 0.800) or other Chinese cities . Similarly, we did not find significant changes in the number of COVID-19 cases associated with decreasing relative humidity in Wuhan  and other Chinese cities .4In this analysis covering 202 locations in 8 countries, meteorological conditions  were not significantly associated with the COVID-19 transmission, suggesting that warmer weather alone seems unlikely to reduce the spread ability of the pandemic. To our knowledge, this is the first study at a global scale to examine the relationship between meteorological conditions and epidemiological characteristics of COVID-19 using multiple regression models, mete-analysis, wavelet analysis and SEIR models.Previous studies reported that certain meteorological factors may have effect on the transmission of respiratory-borne infectious diseases . The conIn this multi-country analysis, we did not observe the aforementioned relationship between temperature and transmission of COVID-19, even after adjusting for other potential confounders. Several prior studies also found that temperature did not play significant role in COVID-19 infection , for exa0 of COVID-19, is influenced by a number of factors such as various lockdown policies across countries, different phases of the COVID-19 epidemic, as well as other unmeasured confounders. In response, we conducted a two-stage analysis  to account for the variation of control policy across countries. Also, to facilitate a comparison of R0 across various locations, we uniformly chose a total of 18 days in each location  to calculate R0. Secondly, our study is ecological in nature, with lack of individual-level  and some location-specific information , which may have an influence on the COVID-19 transmission and confound our results. Another limitation is the use of relative humidity, which is be highly related to temperature and may is not an adequate measure of humidity. Also, we chose these 202 locations because detailed daily data of new confirmed COVID-19 cases and environmental factors of these locations are available in the public database, which may cause selection bias to some extent. Future studies should develop complicated models with higher spatial-temporal resolution to assess the relationship between meteorological conditions and the epidemiological characteristics of COVID-19.Our study has strengths and limitations. The major strength is utilizing data from 202 locations in 8 countries, covering wide climate areas and enabling our findings generalized globally. Also, there are several limitations of this study. Firstly, our major outcome, R5Conclusively, this study provides the first global evidence that meteorological factors do not have significant effect on the COVID-19 transmission. Given the lack of association, public health agencies should not rely on the warm weather to flatten the curve of COVID-19 transmission. Therefore, the non-medication interventions should be implemented consistently to keep constraining the epidemic caused by SARS-CoV-2, in case of another resurgence.10.13039/100000865Bill & Melinda Gates Foundation, Seattle, WA [Grant No. OPP1216424] and the Fudan University Research Project on COVID-19 Emergency, China, SH [Grant No. IDF201007].This work was supported by the The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.The Institutional Review Board at the School of Public Health, Fudan University, approved the study protocol with a waiver of informed consent. Data were analyzed at aggregate level and no participants were contacted.Jinhua Pan: Investigation, Methodology, Formal analysis and Roles/Writing-original draft. Ye Yao: Methodology, Formal analysis, Software and Writing-Review and Editing. Xia Meng: Investigation, Data curation, Visualization and Writing-Review and Editing. Zhixi Liu: Investigation, Software, Visualization and Roles/Writing-original draft. John S Ji: Investigation, Writing-Review and Editing. Yang Qiu: Data curation, Writing-Review and Editing. Weidong Wang: Investigation, Data curation. Lina Zhang: Investigation, Data curation. Weibing Wang: Conceptualization, supervision, funding acquisition and writing-review and editing. Haidong Kan: Conceptualization, supervision, resources and Writing-review and editing. All authors critically reviewed and approved the final version of the manuscript. The corresponding authors are responsible for ensuring that the descriptions are accurate and agreed by all authors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Preeclampsia is a pregnancy-induced disorder that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. HLA-G is thought to play important roles in maternal–fetal immune tolerance, and the associations between HLA-G gene polymorphisms and the onset of pregnancy-related diseases have been explored extensively. Because contiguous genomic sequencing is difficult, the association between the HLA-G genotype and preeclampsia onset is controversial. In this study, genomic sequences of the HLA-G region (5.2 kb) from 31 pairs of mother–offspring genomic DNA samples  were obtained by single-molecule real-time sequencing using the PacBio RS II platform. The HLA-G alleles identified in our cohort matched seven known HLA-G alleles, but we also identified two new HLA-G alleles at the fourth-field resolution and compared them with nucleotide sequences from a public database that consisted of coding sequences that cover the 3.1-kb HLA-G gene span. Intriguingly, a potential association between preeclampsia onset and the poly T stretch within the downstream region of the HLA-G*01:01:01:01 allele was found. Our study suggests that long-read sequencing of HLA-G will provide clues for characterizing HLA-G variants that are involved in the pathophysiology of preeclampsia. Although extensive studies have been conducted, the etiology of PE remains unknown. However, multiple factors have been implicated, including endothelial5, immunological6, and genetic factors8. It has been suggested that an inadequate maternal immunological response to the semiallogenic fetus and consequently abnormal placentation could cause PE. The induction of immune tolerance to the fetus might be critical for the mother, and histocompatibility is expected to play an important role in this immune tolerance.Preeclampsia (PE) is a pregnancy-induced condition that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. Recently, epidemiological studies have suggested that expectant mothers who are affected by PE have a higher risk of cardiovascular diseases, including hypertension, in later life9. Fournel et al. suggested that the soluble form of the HLA-G protein secreted from extravillous trophoblasts could protect the fetus from the mother’s immunity10. Similarly, Fuzzi et al. suggested that only embryos that produce soluble HLA-G can survive after in vitro fertilization11. With regard to the relationship between HLA-G expression and PE, Farina et al. showed that mRNA expression of HLA-G was decreased in PE patients compared with healthy controls12. These studies suggest that soluble HLA-G plays a critical role in the induction of immune tolerance in the fetus.HLA-G, which is expressed in the extravillous trophoblasts of the placenta, is believed to play an important role in the establishment of maternal–fetal immune tolerance, and associations between HLA-G gene polymorphisms and PE have been suggested13. However, several follow-up studies failed to support the findings of Hylenius et al.17. Among those studies, Larsen et al. found that the 14-bp deletion/insertion polymorphism (rs371194629) accompanying other polymorphisms in exon 8  was associated with PE in the primipara when the fetus was homozygous for the insertion18. The results by Larsen et al. indicate the importance of contiguous haplotype data for investigation of the association between HLA-G and PE.The genetic relationship between HLA-G polymorphisms and PE, however, has not been clearly demonstrated. Hylenius et al. reported that paternal inheritance of a 14-bp deletion/insertion in HLA-G can be important for PE onset19 and 3.5KJPN databases (a whole-genome reference panel for the Japanese population20). A solution to the issue of uncertain genotypes at the HLA loci might be long-read sequencing21. The length of the HLA-G locus (5.2 kb) is within the PCR amplification range if appropriate primer sets are designed20. This finding might be useful for identifying unique, nonpolymorphic regions that flank the HLA-G locus. Therefore, the sequencing of PCR amplicons spanning the whole HLA-G locus using long-read sequencers is a feasible approach for population genetic studies of the association between PE and HLA-G variants.Previous HLA-G genotyping studies of patients with PE have exhibited controversial results. Genotyping of HLA genes is quite difficult because of its complex structure. Tadaka et al. discussed obvious discordance in the allele frequencies at the HLA loci between the GnomADIn the present study, we first analyzed full‐length consensus sequences of the HLA-G locus in mother–offspring pairs with complications of PE and healthy pregnancies using single-molecule real-time sequencing on the PacBio RS II instrument. Additionally, we investigated PE-related distributions of HLA-G alleles and the compatibility of HLA-G genotypes in mother–offspring pairs and explored new alleles of the HLA-G locus.https://www.ebi.ac.uk/ipd/imgt/hla/). After filtration according to the number of subreads of a consensus sequence, we obtained one or two consensus sequences for each sample, which likely corresponded to homozygotes or heterozygotes, respectively , and our amplicons covered the entire region. We compared the obtained PacBio consensus sequences with the sequences of known HLA-G alleles by a BLAST search. Most of the consensus sequences were consistent with one of seven major HLA-G alleles; however, eight PacBio consensus sequences were inconsistent with any known HLA-G alleles . Heritability was not confirmed for either new allele in our collection.For the two PacBio consensus sequences that were inconsistent with the known HLA-G sequences, the Sanger sequencing results corresponded to the PacBio consensus data  mother–offspring pairs is shown in Table 24, although the HLA-G alleles of previous studies were limited to second- or third-field resolution, whereas our results were expanded to fourth-field resolution. The distribution of the HLA-G alleles was not significantly different between the PE and control mother–offspring pairs  and 29.8% (37/124) of all determined alleles, respectively. The data were consistent with previous reports that G*01:01:01 and G*01:04 are the most common alleles in the Japanese population/124 and 19, and in the Japanese population, using the Tohoku Medical Megabank Organization (ToMMo-4.7KJPN)25, showed that our frequencies for each SNP and short insertion/deletion (indel) were highly consistent with those large-scale data  and confirmed the accuracy of our sequencing results 18. There was no significant difference in the allelic frequencies of the 14-bp indel between PE and healthy mother–offspring pairs in our cohort  is controversial in terms of its association with PE. This indel is located outside of the well-characterized 3.1 kb HLA-G core region, and the insertion is conserved in the chimpanzee genome . The relationship between HLA-G alleles and the 14-bp indel was evaluated by long-read de novo sequencing using PacBio RS II. The G*01:01:02:01, G*01:01:03:03, G*01:03:01:02, and G*01:06:01:01 alleles contain the 14-bp insertion, whereas the other alleles do not, which is the same as in the human reference genome sequence 26 as well as the 4.7KJPN database. The length of the stretches is 17–43 bp in our collection, 13–38 bp in 4.7KJPN, and 17–50 bp in the WebSTR database. Even though the range of the lengths of the T stretches we observed is within the ranges of the previous studies, we further checked the differences in the T stretch length between mother and offspring alleles that would be shared between the two and found that the differences would be approximately 0.8 to 2.8 among those cases . The length of the poly T stretch in hg19 is 21 bp, and the corresponding length in the chimpanzee genome is 24 bp. The stretch is polymorphic, and multiple alleles have been reported in the WebSTR database  Fig. . In addiUsing single-molecule real-time sequencing on the PacBio RS II instrument, we obtained full‐length consensus sequences of HLA-G loci in 18 PE and 13 healthy mother–offspring pairs. The PacBio long-read sequencing data corresponded well to short-read resequencing population data. In addition, two new alleles of HLA-G were identified in addition to seven well-known HLA-G alleles. Moreover, a long T stretch downstream of HLA-G outside of the commonly characterized 3.1 kb region was found. The lengths of the stretches showed divergence (from 17 to 43 bp). Interestingly, the shorter poly T stretch (< 25 bp) in the HLA-G*01:01:01:01 allele, the most prevalent HLA-G allele, in the offspring appears to be associated with the onset of maternal PE in a dominant manner.29. Based on the data shown in Supplementary Table The PacBio sequencing results corresponded well to those of previous studies. For example, the minor allele frequencies of the HLA-G region were similar to those in the 4.7KJPN and GnomAD EAS populations. This finding suggests that the PCR amplicon sequencing approach can be used together with PacBio sequencing, whose high accuracy is shown in human whole genome sequencing9. However, we did not find any direct genetic evidence that pertained to the function of soluble HLA-G in our collection. A novel allele that contained the stop-gain SNP rs144753960, observed in a PE mother . This study was conducted in accordance with the Declaration of Helsinki. Genomic DNA was extracted from whole peripheral blood (mothers) or umbilical cord blood (offspring) as described in34.Subjects were obtained from the Birth and Three-Generation Cohort Study established by the Tohoku Medical Megabank Project35 to design candidate PCR primer pairs for the HLA-G region after masking the repetitive sequences using RepeatMasker (4.0.7)36. To ensure successful PCR for as many samples as possible, we used a Japanese whole-genome reference panel (3.5KJPN version 2)20 to exclude the PCR primers whose five bases at the 3′ end overlap any polymorphic sites. Finally, we searched for potential off-target annealing sites of the primers using primer BLAST in the nr database . The primer sequences are shown in Supplementary Table We attempted to sequence the whole region of the HLA-G gene, including the promoter region 5.2 kb). In general, HLA genes are rich in polymorphisms and homologous to each other. Therefore, it is difficult to obtain PCR primers for HLA genes that can be applied to all individuals of a large population. We employed Primer 3 software.2 kb. InTo obtain the template DNA fragment used for amplicon sequencing, two-step PCR was performed under the following conditions. Template DNA  was added to 0.75 U PrimeSTAR GXL DNA polymerase , 2 pmol/µl forward and reverse primers . We have seven amplicon libraries per sample, including samples for other projects (data not shown). Eight samples were subjected to long-read sequencing in one SMRT cell , and each amplicon library was adjusted to 0.083 pmol/SMRT cell. To distinguish individuals, we added barcodes during the second PCR step. Adjustment of the library DNA concentration was performed using Binding Calculator ver. 2.3.1.1. PacBio sequencing reactions were conducted using P6-C4 chemistry following the manufacturer’s protocol (Pacific Biosciences).The sequencing data for the HLA-G locus were analyzed using RS_Long_Amplicon_Analysis in SMRT analysis version 2.3.0 software (Pacific Biosciences). Because seven amplicons from eight samples were analyzed in an SMRT cell, we had to distinguish DNA barcodes using the option “Different on each end (paired)”. Consensus reads of the HLA-G amplicons were built using a minimum subread length of 5064 and a maximum number of subreads of 4000 after several trials. To filter out less reliable consensus reads, we employed filtering by setting the number of reads in a consensus to > 84. One consensus sequence that appeared to be a chimera of two known alleles was removed after manual inspection.After comparison with known HLA-G sequences, there were 41 discordant bases among all 124 HLA-G consensus sequences obtained by PacBio. To verify the discrepancies, we employed cycle sequencing to detect single nucleotide differences, including deletions, and 10% polyacrylamide gel electrophoresis to detect longer nucleotide differences found within the 3′ poly T stretch of amplicons of the 01:01:01:01 allele. In both cases, we used the insert libraries from PacBio sequencing as PCR templates. The primers are shown in Supplementary Table 22. A dendrogram of the HLA-G alleles was drawn using ClustalW with the default options in PhyML (https://www.genome.jp/). Other statistical comparisons were performed using Fisher’s exact test. We considered the results to be statistically significant at p < 0.05.A comparison between the known HLA-G alleles and PacBio consensus reads was performed using the BLAST command-line tool with the default optionsSupplementary Information.